The present invention relates to an assay method and a kit for an assay employing a sensor chip for a fluorescent measuring apparatus utilizing a surface plasmon-field enhanced Fluorescence Spectrometry.
A surface plasmon-field enhanced Fluorescence Spectrometry (hereafter, merely referred to as “SPFS”) is a method capable of detecting an extremely small amount and/or extremely low concentration of analytes by efficiently exciting fluorescent dyes to label analytes (analysis target material) captured in the vicinity of a metal film by utilizing plasmon resonance which is increased to some tens of times to some hundreds of times by resonance between surface plasmon and evanescent wave which is transmitted through a metal layer when the surface of the metal layer formed on a dielectric substance is irradiated with exciting light such as laser light on the condition that total-reflection decrement is caused.
A sensor base plate used for such SPFS is described in Patent document 1. Patent document 1 discloses a sensor unit 100 as shown in FIG. 2, and the sensor unit 100 includes a transparent plate made of glass, plastics, or other transparent material; a metal film formed on one side of this plate by sputtering; a dextran layer (dextran film) bonded with this metal film, and ligands (antibodies) bonded with this dextran film. These ligands cause interaction (antigen-antibody reaction) with specific bio molecules (for example, antigens) which exist in a sample liquid, and Patent document 1 teaches about implementation of fluorescence measurement by utilization of SPFS with variable angle inner total reflection fluorescence.
In this connection, in Patent document 1, as mentioned above, analytes are detected using an antigen-antibody reaction. However, in the antigen-antibody reaction described in Patent document 1, there is a problem that immune reaction between antibodies and antigens is not yet enough. Generally, enhancement of the reactivity of the immune reaction between antibodies and antigens enhances assay light emission signal to be detected in proportion to the enhancement, thereby improving the reactivity between antibodies and antigens.
However, in proportion to the improvement of the immune reaction between antibodies and antigens, nonspecific reaction is also enhanced so as to increase assay noise signals which is a noise component. As a result, the measurement sensibility of analytes is not improved. Then, in a method disclosed in Patent document 2, in order to increase the measurement sensibility by suppressing increase of the assay noise signals, antibodies or antigens are mixed with both polyethylene glycols and urea, and antibodies and antigens are made to react with each other under the coexistence of these polyethylene glycols and urea.
Patent document 1: Japanese Patent No. 3294605 official report
Patent document 2: Japanese Unexamined Patent Publication No. 2010-145202 official report
However, it turned out that although an antigen-antibody reaction with a mixture of polyethylene glycols and urea as described in Patent document 2 improves the measurement sensitivity as compared with conventional method, this technique even which is diverted to SPFS is insufficient to realize high accuracy detection for an extremely small amount and/or extremely low concentration of analytes, and the increase of S (assay light emission signal)/N (assay noise signal) ratio (measurement sensitivity) being a ratio of assay light emission signals to assay noise signals is restrictive. Further, as described in Patent document 2, in the embodiment that polyethylene glycol is mixed in the liquid, its concentration is limited. That is, for example, if polyethylene glycol is mixed in a liquid with extremely high concentration, the viscosity of the liquid becomes high too much, so that reactivity is lowered, and S (assay light emission signal) is decreased.